Biomedical subjects

J W Drijfhout

Publications and source records attributed to J W Drijfhout.

At least 37 records · Page 2Linked to original sources

PubMed2000

Antigen arrays in T cell immunology.

The screening of compound arrays in in vitro bioassays has developed into a powerful tool for the identification of biologically active substances. In the past decade, this technology has increasingly been applied to immunology. As the specificity of the immune system is determined by antigen detection via receptors on B and T cells, targeting the specificity of these immune receptors with random arrays is unique in its ability to generate general and quantitative information on cellular (cross-)reactivity. Synthetic array studies have been useful for identification of epitopes and antigens from databases by defining recognition patterns, isolation of synthetic peptides capable of modulating T cell responsiveness, characterisation of TCR promiscuity, and identification of functionally cross-reacting peptides that are potentially involved in molecular mimicry.

Combinatorial Chemistry Techniques↗

PubMed2000

Structure of celiac disease-associated HLA-DQ8 and non-associated HLA-DQ9 alleles in complex with two disease-specific epitopes.

The association of celiac disease (CD) with HLA-DQ2 and HLA-DQ8 is indicative of preferential mucosal T cell recognition of gluten fragments bound to either DQ allele. We have recently identified two gluten-derived, HLA-DQ8-restricted T cell stimulatory peptides, one each from gliadin and glutenin, recognized by specific T cell clones derived from the small intestine of CD patients. We have now performed molecular modeling and examined the fine specificity of these peptides in complex with HLA-DQ8. There is only one binding register for both peptides, with glutamine residues at the p1 and p9 anchor positions. Both T cell clones recognize substituted peptides at p1 and p9, but poorly so at p2-p8, especially the gliadin-specific clone. Contrasting patterns of recognition of p9Gln --> Glu peptide variants (both predicted as better DQ8 binders by modeling) were observed: enhancement of recognition for the gliadin peptide, yet complete absence thereof for the glutenin peptide. The double-substituted gliadin peptide variant p1/9Gln --> Glu, which can also arise by pepsin/acid/transglutaminase treatment, shows a considerable increase in sensitivity of recognition, consistent with better binding of this peptide to DQ8, as predicted by energy minimization. Surprisingly, the two native peptides are also recognized by their respective T cell clones in the context of the related molecule HLA-DQ9 (beta57Asp(+)). The p1/9Gln --> Glu gliadin peptide variant is likewise recognized, albeit with a 10-fold lower sensitivity, the first reported p9Glu binding in a beta57Asp(+) MHC II allele. Our results have important implications for the pathogenesis of autoimmune disease and the possible manipulation of aberrant responses thereof.

Alleles↗

PubMed2000

T-cell lines reactive to an immunodominant epitope of the tyrosine phosphatase-like autoantigen IA-2 in type 1 diabetes.

Type 1 diabetes is the result of destruction of the insulin-secreting beta-cells of the pancreas by a process in which T-cells play a central role. A tyrosine phosphatase-like protein, IA-2, is a major target for autoantibodies and T-cells in the disease. In this study, we have further characterized the T-cell response to IA-2 by the generation and characterization of T-cell lines. T-cell lines responsive to IA-2 antigen were generated from 17 of 32 patients and 3 of 10 control subjects. Antigen specificity was confirmed in lines from six diabetic patients and one control individual by demonstration of responses to synthetic IA-2 peptides and epitope mapping. Five lines from diabetic patients responded to one of two peptides representing amino acids 831-850 and 841-860 of IA-2. The overlapping portion may therefore represent an immunodominant region of the molecule. The sixth patient-derived line responded to a peptide representing amino acids 751-770 of IA-2 presented by the DR 4 (DRB1*0401) allele that confers susceptibility to type 1 diabetes. Primary T-cell responses to peptides of the immunodominant region were detected in 9 of 19 (47%) type 1 diabetic patients and 16 of 22 (73%) nondiabetic siblings, consistent with this region having immunostimulatory properties. The study reports for the first time T-cell lines reactive to IA-2 from diabetic patients and defines an immunodominant region of the molecule.

Adolescent↗

PubMed1999

Fluorogenic MMP activity assay for plasma including MMPs complexed to alpha 2-macroglobulin.

Elevated MMP activities are implicated in tissue degradation in, e.g., arthritis and cancer. The present study was designed to measure MMP enzyme activity in plasma. Free active MMP is unlikely to be present in plasma: upon entering the circulation, active MMP is expected to be captured by the proteinase inhibitor alpha 2-macroglobulin (alpha 2M). Reconstituted MMP-13/alpha 2M complex was unable to degrade collagen (MW 300,000) in contrast to the low-molecular-weight fluorogenic substrate (MW < 1500). Limited access of high-MW substrates to the active site of MMPs captured by alpha 2M presents the most likely explanation. Consistently, the high-MW inhibitor TIMP (MW approximately 28,000) was unable to inhibit MMP/alpha 2M enzyme activity, whereas the low-MW inhibitor BB94 (MW approximately 500) effectively suppressed enzyme activity. By using fluorogenic substrates with Dabcyl/Fluorescein as quencher/fluorophore combin-ation, sensitive MMP-activity assays in plasma were achieved. Spiking of active MMP-13 and MMP-13/alpha 2M complex, and inhibitor studies with TIMP-1 and BB94, indicated that active MMPs are efficiently captured by alpha 2M in plasma. MMP activity was even detected in control plasma, and was significantly increased in plasma from rheumatoid arthritis patients.

Collagen↗

PubMed1999

Identification of a conserved universal Th epitope in HIV-1 reverse transcriptase that is processed and presented to HIV-specific CD4+ T cells by at least four unrelated HLA-DR molecules.

CD4+ Th cells play an important role in the induction and maintenance of specific T cell immunity. Indications for a protective role of CD4+ T cells against HIV-1 infection were found in subjects who were able to control HIV-1 viremia as well as in highly HIV-1-exposed, yet seronegative, individuals. This study describes the identification of an HIV-1-specific Th epitope that exhibits high affinity binding as well as high immunogenicity in the context of at least four different HLA-DR molecules that together cover 50-60% of the Caucasian, Oriental, and Negroid populations. This HIV-1 reverse transcriptase-derived peptide (RT171-190) is highly conserved among different HIV-1 isolates. Importantly, stimulation of PBL cultures from HIV-1 seronegative donors with this peptide resulted in Thl-type lymphocytes capable of efficient recognition of HIV-1-pulsed APCs. Taken together, these data indicate that peptide RT171-190 constitutes an attractive component of vaccines aiming at induction or enhancement of HIV-1-specific T cell immunity.

Amino Acid Sequence↗

PubMed1999

Differential binding of viral peptides to HLA-A2 alleles. Implications for human papillomavirus type 16 E7 peptide-based vaccination against cervical carcinoma.

Several cancer immune intervention protocols aim at inducing T cell immunity against antigens presented by HLA-A2, the most common human MHC class I molecule. In the context of HLA-A*0201, we previously identified two cytotoxic T lymphocyte epitopes (E7(11-20) and E7(86-93)) encoded by the human papillomavirus type 16 E7 (HPV16 E7) oncoprotein, which is a tumor-specific antigen for cervical carcinoma. This study reports that the two HPV16 epitopes and a control hepatitis B virus epitope bind equally well to five HLA-A2 alleles (A*0201, A*0202, A*0203, A*0204, and A*0209). These HLA-A2 variants display comparable binding characteristics in accordance with the A2 supertype (M. F. Del Guercio et al., J. Immunol. 1995. 154: 685-693). Cervical carcinoma patients expressing these alleles may benefit from vaccination with the two HPV16 E7 peptides. In contrast, none of the peptides tested bound to A*0207 or A*0208, whereas heterogeneous binding was observed for A*0205 and A*0206. Therefore, the amino acid substitutions that discriminate these HLA-A2 variants from A*0201 affect antigen presentation. Taken together, our findings have implications for application of the A2 supertype concept and for vaccination with A*0201-binding peptides, in particular HPV16 E7 peptides.

Alleles↗

PubMed1999

Quantitative determination of TCR cross-reactivity using peptide libraries and protein databases.

A single T cell clone can be activated by many different peptides in the context of a particular HLA molecule. To quantify the number of peptides that can be recognized by a CD4(+) T cell clone, we screened a one-bead-one-peptide synthetic peptide library and a protein database for peptides that stimulate an HLA-DR3-restricted, human glutamic acid decarboxylase (GAD65)-reactive CD4(+) T cell clone. Both the library screening and the database analysis indicated that this T cell clone is able to recognize approximately 10(6) 11-mer peptides at low nanomolar concentration. Furthermore, we determined that the frequency of cross-reactivity increased only 1.5-3 times when the peptide concentration increased 10 times, in the range of 0.01 - 1 microM. These data imply that there is a considerable potential for T cell cross-reactivity and are useful for studies on the role of molecular mimicry in the etiology of T cell-mediated disease.

Amino Acid Sequence↗

PubMed1999

Glutenin is involved in the gluten-driven mucosal T cell response.

Gluten ingestion causes coeliac disease in susceptible individuals. Gluten is a heterogeneous mixture of glutenin and gliadin, the latter of which is considered responsible for disease induction. By combining high-performance liquid chromatography purification steps of gluten with a T cell bioassay and mass spectral analyses, we have identified a glutenin peptide (glt04 707-742) that activates T cells from the small intestine of a coeliac disease patient and results in the secretion of large amounts of IFN-gamma. The minimal T cell stimulatory core of the peptide (residues 724-734) is repetitively present in glutenin molecules. Moreover, it was observed that a large number of naturally occurring variants of this peptide are recognized by the T cells. These data suggest that the large heterogeneity of glutenin proteins dramatically increases the number of available T cell epitopes. Together, the results provide new insight into the nature of the gluten antigens that lead to coeliac disease and suggest that glutenin, next to gliadin-derived antigens, may be involved in the disease process.

Amino Acid Sequence↗

PubMed1999

Limitations of homology searching for identification of T-cell antigens with library derived mimicry epitopes.

Mimicry epitopes that are recognized by T-cells can be identified through screening of synthetic peptide libraries. We have shown that these mimicry epitopes share sequence similarity with the corresponding natural epitopes and that mimicry sequences can be used for the definition of protein derived T-cell epitopes from databases. This can be done by either homology searching or pattern searching. Here we discuss the advantages and disadvantages of homology searching as an alternative for the generally applicable recognition pattern approach. We show that only for part of the library derived mimicry epitopes, the degree of similarity to the natural epitope may be high enough for successful homology searching in small databases.

Amino Acid Sequence↗

PubMed1999

Definition of agonists and design of antagonists for alloreactive T cell clones using synthetic peptide libraries.

Alloreactive T cells form an important barrier for organ transplantation. To reduce the risk of rejection patients are given immunosuppressive drugs, which increase the chance of infection and the incidence of malignancies. It has been shown that a large proportion of alloreactive T cells specifically recognize peptides present in the groove of the allogeneic MHC molecule. This implies that it might be possible to modulate the alloresponse by peptides with antagonistic properties, thus preventing rejection without the side effects of general immunosuppression. Peptide antagonists can be designed on the basis of the original agonist, yet for alloreactive T cells these agonists are usually unknown. In this study we have used a dedicated synthetic peptide library to identify agonists for HLA-DR3-specific alloreactive T cell clones. Based on these agonists, altered peptide ligands (APL) were designed. Three APL could antagonize an alloreactive T cell clone in its response against the library-derived agonist as well as in its response against the original allodeterminant, HLA-DR3. This demonstrates that peptide libraries can be used to design antagonists for alloreactive T cells without knowledge about the nature of the actual allostimulatory peptide. Since the most potent agonists are selected, this strategy permits detection of potent antagonists. The results, however, also suggest that the degree of peptide dependency of alloreactive T cell clones may dictate whether a peptide antagonist can be found for such clones. Whether peptide antagonists will be valuable in the development of donor-patient-specific immunosuppression may therefore depend on the specificity of the in vivo-generated alloreactive T cells.

Amino Acid Sequence↗

PubMed1999

Get into the groove! Targeting antigens to MHC class II.

The activation of MHC class II-restricted helper T cells is paramount to adaptive immune responses. Vaccine development could therefore benefit from improved ways of targeting antigens into MHC class II molecules. In recent years, the natural pathways of MHC class II antigen presentation have been exploited to achieve this goal. First, antigenic proteins and peptides have been modified to facilitate receptor-mediated uptake by professional antigen-presenting cells. Second, DNA constructs containing specific targeting sequences have been used to direct endogenously synthesized antigens to the MHC class II compartments. Both strategies proved to be highly effective. We review these data and describe how this knowledge is currently applied to the design of vaccines that activate helper T cells in vivo.

Amino Acid Sequence↗

PubMed1998

Definition of natural T cell antigens with mimicry epitopes obtained from dedicated synthetic peptide libraries.

Progress has recently been made in the use of synthetic peptide libraries for the identification of T cell-stimulating ligands. T cell epitopes identified from synthetic libraries are mimics of natural epitopes. Here we show how the mimicry epitopes obtained from synthetic peptide libraries enable unambiguous identification of natural T cell Ags. Synthetic peptide libraries were screened with Mycobacterium tuberculosis-reactive and -autoreactive T cell clones. In two cases, database homology searches with mimicry epitopes isolated from a dedicated synthetic peptide library allowed immediate identification of the natural antigenic protein. In two other cases, an amino acid pattern that reflected the epitope requirements of the T cell was determined by substitution and omission mixture analysis. Subsequently, the natural Ag was identified from databases using this refined pattern. This approach opens new perspectives for rapid and reliable Ag definition, representing a feasible alternative to the biochemical and genetic approaches described thus far.

Antigens, Bacterial↗

PubMed1998

Small intestinal T cells of celiac disease patients recognize a natural pepsin fragment of gliadin.

Celiac disease is a common severe intestinal disease resulting from intolerance to dietary wheat gluten and related proteins. The large majority of patients expresses the HLA-DQ2 and/or DQ8 molecules, and gluten-specific HLA-DQ-restricted T cells have been found at the site of the lesion in the gut. The nature of peptides that are recognized by such T cells, however, has been unclear so far. We now report the identification of a gliadin-derived epitope that dominantly is recognized by intestinal gluten-specific HLA-DQ8-restricted T cells. The characterization of such epitopes is a key step toward the development of strategies to interfere in mechanisms involved in the pathogenesis of celiac disease.

Amino Acid Sequence↗

PubMed1998

Purification of toxic compounds from larvae of the gray fleshfly: the identification of paralysins.

Larval haemolymph of Neobellieria bullata (Insecta, Diptera) is highly toxic to adults of the same species: injection causes instant paralysis to death. Referring to their dramatic effect in adult insects the responsible compounds were designated paralysins. Two paralysins, soluble in organic solvents and heat stable, were chromatographically purified to homogeneity. They were identified by use of mass spectrometry and nuclear magnetic resonance respectively as beta-alanine-tyrosine (beta-Ala-Tyr) and as 3-hydroxy-kynurenine (3-HK). The quantities of beta-Ala-Tyr and 3-HK in the insect appear to increase steadily during larval development, with peak values prior to the pupal stage. These findings may contribute to a better understanding of some aspects of the process of insect metamorphosis. Orienting experiments in mammals suggest that both compounds, when injected intraspinally, are also neurotoxic to rats. In addition, cytotoxicity tests revealed that 3-HK, but not beta-Ala-Tyr is toxic to human neuroblastoma cells, rat primary cortex neurons as well as to rat glial cells.

Animals↗

PubMed1998

Enhancement of tumor outgrowth through CTL tolerization after peptide vaccination is avoided by peptide presentation on dendritic cells.

Synthetic peptide-based vaccines have been shown to induce potent protective and therapeutic T cell-mediated immunity in preclinical animal models and are now being evaluated in clinical phase I/II studies for their efficacy against tumors or infectious diseases. However, such vaccines might also specifically tolerize T cells causing enhanced tumor outgrowth, as shown by vaccination with two CTL epitopes derived from the adenovirus type 5 early region 1 (Ad5E1) oncogenes. We now report that modification of the Ad5E1 peptide vaccine either through incorporation of the peptides into liposomes or by ligation of the peptides to lipid tails, another vaccine formulation being tested in the clinic, fails to convert immunosuppression into effective antitumor vaccination. Inclusion of a helper T cell epitope into the vaccine likewise induces enhanced tumor outgrowth and thus does not diminish the capacity of the peptides to tolerize Ad5E1-specific CTL. In contrast, the Ad5E1-derived peptides evoke a strong tumor-protective CTL response when presented on dendritic cells (DC), indicating that the in vivo CTL-tolerizing potential of these peptides is converted to specific immunostimulation when presented on DC. These findings have important implications for the development of peptide-based immune intervention strategies and emphasize the superior nature of Ag-pulsed DC over other peptide-based vaccination protocols as well as the crucial importance of the mode of peptide-Ag delivery in setting the balance between T cell stimulation and tolerization.

Amino Acid Sequence↗

PubMed1998

Immunization with human papillomavirus type 16 (HPV16) oncoprotein-loaded dendritic cells as well as protein in adjuvant induces MHC class I-restricted protection to HPV16-induced tumor cells.

Human papillomavirus (HPV) E6 and E7 oncoproteins are attractive targets for T-cell-based immunotherapy of cervical cancer. In this study, we demonstrate that dendritic cells (DCs) pulsed with HPV16 E7 protein are not only recognized in vitro by E7-specific CTLs but also elicit E7-specific CTL responses in vivo, associated with protection against a challenge with syngeneic HPV16-induced tumor cells. Vaccination with soluble E7 protein in incomplete Freund's adjuvant likewise induces E7-specific CTL responses associated with tumor protection. The presence of HPV16 E7-specific CTLs in vivo and the observation that depletion of CD8+ cells completely abolishes tumor protection demonstrate that CTLs are the major effector cells in mediating antitumor activity. The in vivo involvement of DCs in the activation of protective CTLs is suggested by the surface display of E7 peptide-loaded MHC class I molecules on these cells after E7 protein immunization. These data show that HPV16 E7 protein-pulsed DCs, as well as the administration of E7 protein antigen in adjuvant, can effectively stimulate tumor-specific MHC class I-restricted CD8+ T-cell-mediated protective immunity to HPV16-induced cancers.

Animals↗

PubMed1998

Simultaneous humoral and cellular immune response against cancer-testis antigen NY-ESO-1: definition of human histocompatibility leukocyte antigen (HLA)-A2-binding peptide epitopes.

A growing number of human tumor antigens have been described that can be recognized by cytotoxic T lymphocytes (CTLs) in a major histocompatibility complex (MHC) class I-restricted fashion. Serological screening of cDNA expression libraries, SEREX, has recently been shown to provide another route for defining immunogenic human tumor antigens. The detection of antibody responses against known CTL-defined tumor antigens, e.g., MAGE-1 and tyrosinase, raised the question whether antibody and CTL responses against a defined tumor antigen can occur simultaneously in a single patient. In this paper, we report on a melanoma patient with a high-titer antibody response against the "cancer-testis" antigen NY-ESO-1. Concurrently, a strong MHC class I-restricted CTL reactivity against the autologous NY-ESO-1-positive tumor cell line was found. A stable CTL line (NW38-IVS-1) was established from this patient that reacted with autologous melanoma cells and with allogeneic human histocompatibility leukocyte antigen (HLA)-A2(-), NY-ESO-1-positive, but not NY-ESO-1-negative, melanoma cells. Screening of NY-ESO-1 transfectants with NW38-IVS-1 revealed NY-ESO-1 as the relevant CTL target presented by HLA-A2. Computer calculation identified 26 peptides with HLA-A2-binding motifs encoded by NY-ESO-1. Of these, three peptides were efficiently recognized by NW38-IVS-1. Thus, we show that antigen-specific humoral and cellular immune responses against human tumor antigens may occur simultaneously. In addition, our analysis provides a general strategy for identifying the CTL-recognizing peptides of tumor antigens initially defined by autologous antibody.

Aged↗

PubMed1998

A new hybrid resin for stepwise screening of peptide libraries combined with single bead Edman sequencing.

A library system was developed for the discovery of bioactive peptides. Library synthesis and peptide sequencing was performed on a solid support while the screening for bioactivity was done with peptides in solution. The peptides were synthesized by split and mix, one-bead-one-peptide library synthesis, using a Tentagel S-NH2 solid support with a loading of approximately 100 pmol/bead. The major part of the peptide was connected to the support by a single acid-labile linker and a minor part of the peptide was acid-stabile attached to the polymer. The percentage of acid-stabile attached peptides could easily be controlled during modification of the amino functionalities of the resin at the start of the process. The cleavage rate of the acid-labile attached peptide from the resin depends on the composition of the cleavage mixture. When cleavage conditions were carefully controlled, a three-step partial cleavage protocol allowed for convergent bioactivity screening on peptide libraries using only one type of acid-labile linker. The partial cleavage and convergent screening procedure was repeated three times, after which the bead containing the bioactive peptide was sequenced. As such a bead still contained acid-stabile attached peptide, the Edman sequencing was straightforward and repetitive yields were excellent because the immobilized peptide was not washed out.

CD4-Positive T-Lymphocytes↗